(1) Field of the invention
The invention relates to a polypeptide of calcitonin analogue and use thereof.
The polypeptide can be used to prevent and/or cure calcium metabolic diseases such as reduction of ache in osteoporosis, prevention of fracture in osteogenesis imperfecta, and curing of hypercalcemia and Paget's disease.
(2) Related arts
Calcitonin was firstly found by D. H. Copp et al. in the year of 1961 from human jugular vein blood as one of peptide hormones, which shows an activity of reducing a calcium concentration in blood.
The calcitonin has been constructed from 32 amino acid residues having a sequence (substantially corresponding to SEQ ID NO: 1), as shown below. ##STR1##
In the amino acid sequence, Pro.multidot.NH.sub.2 means proline amide residue and Cys residues in the 1st and 7th positions are linked by a disulfide bond.
Thereafter, peptide hormones having biological activities similar to the calcitonin have been extracted from various animals such as salmon, eel, chicken, pig, rat, sheep, cow, rabbit and stingray, and amino acid sequences of such native calcitonins have been determined.
Structural features of calcitonins include a constant chain length of 32 amino acids, a disulfide bridge between the cysteine residues in positions 1 and 7, forming a ring of seven amino acid residues at the N-terminal, and a carboxy terminal proline amide. However, amino acid residues common to all calcitonins are those in 1, 4th-7th, 28th and 32nd positions, only (7 amino acid residues in total). It has been considered that the proline amide at C-terminal, common to all calcitonins, is indispensable for developing biological activities thereof [J. T. Potts, Jr. et al., "Calcium, Parathyroid Hormone and the Calcitonins", page 121 printed by Excerpta Medica, Amsterdam (1971); P. Sieber, "Calcitonin 1969", page 28, Proc. 2nd Symp., printed by Medical Books, London (1970); and W. Rittel et al., "Experientia", Vol. 32, page 246 (1976)].
It has been known that the calcitonins show biological activities of reducing calcium concentration in blood [P. F. Hirsch et al., "Science", Vol. 146, page 412 (1963)], inhibition of feeding [W. J. Freed et al., "Science", Vol. 206, page 850 (1979)], and inhibition of gastric secretion [R. D. Hesch et al., "Horm. Metab. Res.", Vol. 3, page 140 (1971)].
For obtaining the calcitonins, extraction from mammalian blood and tissue samples was firstly tried and then chemical synthesis has been employed. The former has disadvantages of limitation from availability of the raw material and troublesome in purifying operations, and the latter has also disadvantages of that synthetic operations are troublesome, since all of calcitonins are constructed from 32 amino acid residues, and that long period of time is required for synthesis per se and purification, so that it has been quite difficult to provide the calcitonins in a large amount with reasonable cost.
In the recent years, the genetic engineering has remarkably developed and such a process utilizing techniques in the genetic engineering has been proposed, which comprises steps of synthesizing a single-stranded DNA fragment encoding one of calcitonins by using a DNA synthesizer and its single-stranded cDNA, preparing a double-stranded DNA fragment from the single-stranded DNA fragments, purifying the double-stranded DNA fragment, inserting the purified double-stranded DNA fragment into a vector, transforming Escherichia coli by the recombinant vector, cultivating the transformant to express a polypeptide encoding the calcitonin, and separating and purifying the expressed polypeptide. In this case, however, a polypeptide with a C-terminal proline amide and showing the desired biological activities can not be expressed by Escherichia coli and thus the C-terminal amidating treatment is required during the separating-purifying step, but a C-terminal amidating enzyme is expensive, the enzymatic treatment requires special techniques and a skill, and the enzymatic process makes yield of the objective substance low. Therefore, this conventional process can not be said as one suitable for industrial scale production of calcitonins, from view points of cost on the reagent and yield.
Therefore, researchers in the assignee company have energetically studied and investigated on a process utilizing genetic recombination techniques to find that various calcitonin analogues can be obtained without use of the expensive C-terminal amidating enzyme, each of which analogues includes no methionine residue (Met) in its amino acid sequence thereof and C-terminal structure is proline.sup.32 -homoserine.sup.33 amide (Pro.sup.32 -Hse.sup.33 -NH.sub.2), a primary alkyl, 1-20 carbon atoms, amide of homoserine.sup.33 , or an optional polypeptide chain and containing homoserine amide at C-terminal [Jap. Pat. Hei 4 (A. D. 1992) - 59795(A) which corresponds to EP-A-0 464 549]. The process described in these patent literatures is somewhat similar to a process proposed by the assignee company on motilin analogues as one of peptide hormones and disclosed in Jap. Pat. Hei 3 (A.D. 1991) - 80096(A) which corresponds to EP-A-0 355 720. Namely, according to the process described in the patent literatures, each of the calcitonin analogues can be prepared by synthesizing a single-stranded DNA fragment consisting of a leader polypeptide at N-terminus and with Met residue at its C-terminus, a polypeptide encoding the calcitonin analogue gene and with Met residue at its C-terminus, a spacer peptide with Met residue at its C-terminus, a polypeptide encoding the calcitonin analogue gene with Met residue at its C-terminus (the spacer peptide and calcitonin analogue gene being arranged in plural); synthesizing a single-stranded DNA fragment complementary to the first single-stranded DNA fragment; preparing a double-stranded DNA fragment by the single-stranded fragments; inserting the double-stranded fragment into a vector such as a plasmid to obtain a recombinant vector; transforming by the recombinant vector a microorganism such as Escherichia coli or mammalian cell such as CttO cell; cultivating the transformant to express the polypeptide as an inclusion body or fused protein in the transformant; treating the inclusion body with cyanogen bromide to cleave at each position of Met and modify the amino acid residue of Met at C-terminal into Hse or Hse-lactone residue; treating the reaction solution with an acid, for instance 0.1N HCl solution (at 30.degree. C. for 3 hours) to convert all of individual polypeptides into those with Hse-lactone residue at C-terminus; lyophilizing the same; and then treating the same with ammonia solution or reacted with a primary alkyl amine.
The method disclosed in said patent literatures makes possible the preparation of various calcitonin analogues in a large amount and with reasonable cost, but a biological activities of those, for instance salmon I calcitonin-Hse.sup.33 -NH.sub.2 is substantially same with the native Salmon I calcitonin.